Epidermal growth factor stimulates the synthesis of its own receptor in a human breast cancer cell line

The MDA 468 human breast carcinoma cell line was examined for changes in epidermal growth factor (EGF) receptor synthesis and degradation under the influence of EGF. This cell line was used because it overexpresses the EGF receptor such that each cell has 10(6) receptors, but unlike the well-studied A431 cell, its receptor gene is amplified but is not rearranged. On exposure to EGF, total cellular receptor protein, measured by immunoprecipitation with monoclonal antibody B1D8, is reduced. The half-life of receptor metabolically labeled with L-[35S]methionine is 24 h in the absence of EGF and is reduced to 12 h in the presence of 10(-9) M EGF. To measure the effect of EGF on synthesis of the receptor, pulse labeling conditions were selected in which the rate of synthesis of the receptor precursor were followed. EGF had no significant effect on the rate of general protein synthesis in these cells, yet stimulated the synthesis of the EGF receptor 1.8-fold over the unstimulated rate. This increase in receptor precursor synthesis showed time and dose dependence. Stimulation could be detected after 3 h exposure to EGF with a maximum at 6-8 h. A concentration of 10(-11) M EGF gave detectable stimulation with maximal stimulation occurring at 10(-9) M. Longer times and higher concentrations gave submaximal stimulation. A similar dose-response relationship was observed when the rate of mature 170-kDa receptor protein synthesis was measured. These studies demonstrate that EGF stimulates the synthesis of it own receptor. Downregulation of the receptor by EGF results from an increased rate of receptor degradation and not decreased synthesis.     

Phorbol ester or epidermal growth factor (EGF) stimulates the concurrent accumulation of mRNA for the EGF receptor and its ligand transforming growth factor-alpha in a breast cancer cell line

We have previously reported that both 12-O-tetradecanoylphorbol-13-acetate (TPA) and epidermal growth factor (EGF) can stimulate the synthesis rate of EGF receptors. We now show that the MDA468 breast cancer cells express the mRNA for the EGF-like molecule, transforming growth factor-alpha (TGF-alpha), and demonstrate that TPA or EGF cause an accumulation of both EGF receptor and TGF-alpha mRNA. The levels of EGF receptor mRNA paralleled our earlier protein data, with peak accumulations of 2-3-fold with 10(-9) M EGF and 3-5-fold with 100 ng/ml TPA seen between 6 and 8 h. A 7-fold accumulation of TGF-alpha mRNA was seen following 4 h of treatment with TPA, and a 2-fold accumulation was seen after 8 h with EGF. These changes in EGF receptor and TGF-alpha mRNAs were observed in the absence of any change in the mRNA level of the alpha-subunit of hexosaminidase A (a lysosomal enzyme), demonstrating some degree of specificity. Detectable quantities of immunoreactive TGF-alpha accumulated in the cell culture medium of MDA468 cell treated with the blocking anti-EGF receptor monoclonal antibody B1D8 while no immunoreactive TGF-alpha was detected in the medium of cells with unblocked receptors. The concentration of B1D8 used was sufficient to block the binding of exogenously added 125I-EGF to undetectable levels but had only minor effects on cell growth and no effect on the expression of the TGF-alpha and EGF receptor mRNA.     

Receptor-mediated endocytosis of epidermal growth factor by rat hepatocytes: receptor pathway

Substantial amounts of epidermal growth factor (EGF) are cleared from the circulation by hepatocytes via receptor-mediated endocytosis and subsequently degraded within lysosomes. We have used a combined biochemical and morphological approach to examine the fate of the receptor after exposure to EGF. Polyclonal antibodies were prepared against the purified receptor and their specificity established by immunoprecipitation and immunoblotting techniques. The EGF receptor was then localized by immunofluorescence and immunoperoxidase techniques and quantified on immunoblots. In untreated livers, EGF receptor was restricted to the sinusoidal and lateral surfaces of hepatocytes. 2-4 min after exposure of cells to EGF, the receptor was found in small vesicles (i.e., coated vesicles) as well as larger vesicles and tubules at the cell periphery. By 15 min the receptor was found in multivesicular endosomes located near bile canaliculi. Exposure of hepatocytes to EGF also resulted in a rapid loss of receptor protein from total liver homogenates and a decrease in its half-life from 8.7 h in control livers to 2.5 h. This EGF-induced loss of receptors was not observed when lysosomal proteinases were inhibited by leupeptin or when endosome/lysosome fusion was prevented by low temperature (16 degrees C). In the presence of leupeptin, receptor could be detected in structures identified as lysosomes using acid-phosphatase cytochemistry. All these results suggested rapid internalization of EGF receptors in response to ligand and degradation within lysosomes. However, four times more ligand was degraded at 8 h than the number of high-affinity (Kd of 8-15 nM) EGF-binding sites lost, suggesting either (a) high-affinity receptors were recycled, and/or (b) more than 300,000 receptors were available for EGF uptake. We identified and characterized a latent pool of approximately 300,000 low-affinity receptors (Kd approximately 200 nM) that could be separated on sucrose gradients from the plasma membrane pool of approximately 300,000 high-affinity receptors (Kd of 8-15 nM). Despite the differences in their binding affinities, the high- and low-affinity receptors appeared to be structurally identical and were both EGF-dependent protein kinases. In addition, the dynamics of the low-affinity receptors were consistent with a functional role in EGF uptake and delivery to lysosomes.